Background:The central carbon metabolism is important for bacterial survival and fitness. Results: Isotopologue experiments identified amino acid biosynthesis pathways of pneumococci.
Conclusion:The analysis of the carbon metabolism in pneumococci identified an unconventional pathway to synthesize serine. Significance: Understanding the metabolism of pathogens is critical to gain insights into the adaptation strategies and to identify new drug targets.
The genomic analysis of Streptococcus pneumoniae predicted six putative glutamine uptake systems, which are expressed under in vitro conditions, as shown here by reverse transcription-PCR. Four of these operons consist of glnHPQ, while two lack glnH, which encodes a soluble glutamine-binding protein. Here, we studied the impact of two of these glutamine ATP-binding cassette transporters on S. pneumoniae D39 virulence and phagocytosis, which consist of GlnQ and a translationally fused protein of GlnH and GlnP. Mice infected intranasally with D39⌬gln0411/0412 showed significantly increased survival times and a significant delay in the development of pneumococcal pneumonia compared to those infected with D39, as observed in real time using bioluminescent pneumococci. In a mouse sepsis model, the mutant D39⌬gln0411/0412 showed only moderate but significant attenuation. In contrast, the D39⌬gln1098/1099 knockout strain was massively attenuated in the pneumonia and septicemia mouse infection model. To cause pneumonia or sepsis with D39⌬gln1098/1099, infection doses 100-to 10,000-fold higher than those used for wild-type strain D39 were required. In an experimental mouse meningitis model, D39⌬gln1098/1099 produced decreased levels of white blood cells in cerebrospinal fluid and showed decreased numbers of bacteria in the bloodstream compared to D39 and D39⌬gln0411/0412. Phagocytosis experiments revealed significantly decreased intracellular survival rates of mutants D39⌬gln1098/1099 and D39⌬gln0411/0412 compared to wild-type D39, suggesting that the deficiency of Gln uptake systems impairs resistance to oxidative stress. Taken together, our results demonstrate that both glutamine uptake systems are required for full virulence of pneumococci but exhibit different impacts on the pathogenesis of pneumococci under in vivo conditions.
Summary The pneumococcal cell surface protein PavA is a virulence factor associated with adherence and invasion in vitro. In this study we show in vivo that PavA is necessary for Streptococcus pneumoniae D39 colonization of the murine upper respiratory tract in a long-term carriage model, with PavA-deficient pneumococci being quickly cleared from nasopharyngeal tissue. In a pneumonia model, pavA mutants were not cleared from the lungs of infected mice and persisted to cause chronic infection, whereas wild-type pneumococci caused systemic infection. Hence, under the experimental conditions, PavA-deficient pneumococci appeared to be unable to seed from lung tissue into blood, although they survived in blood when administered intravenously. In a meningitis model of infection, levels of PavA-deficient pneumococci in blood and brain following intercisternal injection were significantly lower than wild type. Taken collectively these results suggest that PavA is involved in successful colonization of mucosal surfaces and in translocation of pneumococci across host barriers. Pneumococcal sepsis is a major cause of mortality worldwide so identification of factors such as PavA that are necessary for carriage and for translocation from tissue to blood is of clinical and therapeutic importance.
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